Limited Anti-Inflammatory Role for Interleukin-1 Receptor Like 1 (ST2) in the Host Response to Murine Postinfluenza Pneumococcal Pneumonia

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2 ^−/−) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2^−/− mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1β, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2^−/− mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.     

Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 ^-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 ^-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 ^-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 ^-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 ^-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 ^-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.     

Deletion of the Complement C5a Receptor Alleviates the Severity of Acute Pneumococcal Otitis Media following Influenza A Virus Infection in Mice

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa^−/−) or factor B (Bf ^−/−) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.     

The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2 ^−/− AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2 ^−/− mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.     

Interleukin-1 Receptor–Associated Kinase M–Deficient Mice Demonstrate an Improved Host Defense during Gram-negative Pneumonia

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)–associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M^−/−) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M^−/− alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M^−/− mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M^−/− mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

TNF Drives Monocyte Dysfunction with Age and Results in Impaired Anti-pneumococcal Immunity

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C⁺ monocytes from old (18–22 mo) mice and CD14⁺CD16⁺ intermediate/inflammatory monocytes from older adults also contributed to this “age-associated inflammation” as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C⁺ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C⁺ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C⁺ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity.     

The sst1 Resistance Locus Regulates Evasion of Type I Interferon Signaling by Chlamydia pneumoniae as a Disease Tolerance Mechanism

The sst1, “supersusceptibility to tuberculosis,” locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1^S) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1^S mice. Infection of sst1^S macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-β and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-β. Intriguingly macrophages from the sst1^S mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

TLR2 Signaling Decreases Transmission of Streptococcus pneumoniae by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model

While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of Streptococcus pneumoniae (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of S. pneumoniae from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted S. pneumoniae more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon α and higher viral titers. The greater viral burden in tlr2^−/− mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.     

Hematopoietic but Not Endothelial Cell MyD88 Contributes to Host Defense during Gram-negative Pneumonia Derived Sepsis

Klebsiella pneumoniae is an important cause of sepsis. The common Toll-like receptor adapter myeloid differentiation primary response gene (MyD)88 is crucial for host defense against Klebsiella. Here we investigated the role of MyD88 in myeloid and endothelial cells during Klebsiella pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-Myd88^−/−) and myeloid plus endothelial (Tie2-Myd88^−/−) cells showed enhanced lethality and bacterial growth. Tie2-Myd88^−/− mice reconstituted with control bone marrow, representing mice with a selective MyD88 deficiency in endothelial cells, showed an unremarkable antibacterial defense. Myeloid or endothelial cell MyD88 deficiency did not impact on lung pathology or distant organ injury during late stage sepsis, while LysM-Myd88^−/− mice demonstrated a strongly attenuated inflammatory response in the airways early after infection. These data suggest that myeloid but not endothelial MyD88 is important for host defense during gram-negative pneumonia derived sepsis.     

Mannose-Binding Lectin Regulates Host Resistance and Pathology during Experimental Infection with Trypanosoma cruzi

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas’ disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. In this study we employed MBL^−/− mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas’ disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL^−/− mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL^−/− mice. Importantly, MBL^−/− mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.     

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b⁺ F4/80⁺, CD11b⁺ Ly6C^int Ly6G⁺ and CD11b⁺ Ly6C^hi Ly6G⁻ cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.     

No Impairment in Host Defense against Streptococcus pneumoniae in Obese CPEfat/fat Mice

In the US and globally, dramatic increases in the prevalence of adult and childhood obesity have been reported during the last 30 years. In addition to cardiovascular disease, type II diabetes, and liver disease, obesity has recently been recognized as an important risk factor for influenza pneumonia. During the influenza pandemic of 2009, obese individuals experienced a greater severity of illness from the H1N1 virus. In addition, obese mice have also been shown to exhibit increased lethality and aberrant pulmonary inflammatory responses following influenza infection. In contrast to influenza, the impact of obesity on bacterial pneumonia in human patients is controversial. In this report, we compared the responses of lean WT and obese CPE^fat/fat mice following an intratracheal infection with Streptococcus pneumoniae, the leading cause of community-acquired pneumonia. At 16 weeks of age, CPE^fat/fat mice develop severe obesity, hyperglycemia, elevated serum triglycerides and leptin, and increased blood neutrophil counts. There were no differences between lean WT and obese CPE^fat/fat mice in survival or lung and spleen bacterial burdens following intratracheal infection with S. pneumoniae. Besides a modest increase in TNF-α levels and increased peripheral blood neutrophil counts in CPE^fat/fat mice, there were not differences in lung or serum cytokines after infection. These results suggest that obesity, accompanied by hyperglycemia and modestly elevated triglycerides, at least in the case of CPE^fat/fat mice, does not impair innate immunity against pneumococcal pneumonia.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high¹. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.